1.1 Field of the Invention
The present invention relates generally to the fields of immunology and oncology. Disclosed are compositions comprising nucleotide sequences encoding endonuclease SR, and polypeptides encoded thereby. Also disclosed are methods for repairing DNA and modulating genetic recombination in a cell, particularly through the use of a specific endonuclease, which is referred to hereinafter as Endo-SR (Endonuclease implicated in Switch Recombination). The compositions disclosed are useful in cleaving DNA at specific G-rich regions which are implicated in modulating DNA rearrangements. Also disclosed are methods for the design and isolation of peptidomimetics and Endo-SR inhibitors useful in the treatment of leukemias, lymphomas, and other cancers, as well as modulation of apoptosis and programmed cell death events.
1.2 Description of Related Art
1.2.1 B-Lymphocytes
Mature B lymphocytes can alter their Ig isotype expression by targeted deletional rearrangement of their IgH constant region genes in a process called isotype switch recombination (Harriman et al., 1993; Shimizu and Honjo, 1984). The ability of B cells to xe2x80x9cswitchxe2x80x9d one Ig constant region for another significantly enhances the versatility of the immune system by allowing activated B cells to selectively alter the function of their secreted Ig without altering their ligand specificity (Esser and Radbruch, 1990; Mond et al., 1995a,b).
Recombination breakpoints generated by this process primarily map to large (approximately 1 to 10 kb), highly-repetitive DNA regions found upstream of all IgH constant region genes (Esser and Radbruch, 1990; Harriman et al., 1993). These switch regions are primarily composed of degenerate, variable-length G-rich repeat units, that differ considerably both within and between species (Esser and Radbruch, 1990; Harriman et al., 1993). However, all switch regions contain disproportionate numbers of two pentamer motifs, TGGGN and TGAGC, that are found at or adjacent to most analyzed switch recombination breakpoints (Dunnick et al., 1993; Mowatt et al., 1986; Kenter et al., 1993, Petrini and Dunnick, 1989; Wuerffel et al., 1992).
Selective recombination of individual switch regions directly correlates with transcriptional activation of these regions in response to extracellular signals (Berton and Vitetta, 1990; Bottaro et al., 1994; Kuwabara et al., 1995; Rothman et al., 1990; Warren and Berton, 1995; Xu et al., 1993; Xu and Stavnezer, 1992). Recent studies have demonstrated that an additional signal(s) is required to induce switch recombination at an actively transcribed switch region (Bottaro et al., 1994). Specific transcriptional induction has thus been proposed to increase the accessibility of a particular switch region to switch recombinase factors, which may also be activated by some of the same signals responsible for transcriptional activation of these loci (Mandler et al, 1993; Purkerson and Isakson, 1992; Snapper and Mond, 1993).
The search for switch recombinase factors has primarily focused on the detection of DNA binding factors which specifically interact with switch repeat sequences, and although this approach has lead to the characterization of several novel DNA binding factors (Fukita et al., 1993; Marcu et al., 1992; Miranda et al., 1995; Mizuta et al., 1993; Waters et al., 1989; Wuerffel et al., 1990; Xu et al., 1992), as yet none of these factors have been directly implicated in the switch recombination reaction.
The present invention overcomes limitations in the prior art by providing novel endonuclease polypeptides, and in particular, mammalian Endo-SR polypeptides which have specific endonuclease activity. In a preferred embodiment, the Endo-SR polypeptide is isolated from mammalian sources, including murine, bovine, and human sources, or from organisms such as C. elegans and the like, and comprises at least a 19-contiguous amino acid sequence from SEQ ID NO:2. More preferably, the polypeptide comprises the amino acid sequence of SEQ ID NO:2, and is encoded by a polynucleotide that comprises at least a 76-contiguous nucleic acid sequence from SEQ ID NO:1, and preferably comprising the sequence of SEQ ID NO: 1. Exemplary murine genomic DNA sequences include sequences such as those in SEQ ID NO:3 and SEQ ID NO:4. Preferred sequences include sequences comprising at least 49 contiguous nucleic acid sequences from SEQ ID NO:3, and sequences comprising at least 24 contiguous nucleic acid sequences from SEQ ID NO:4. Sequences comprising at least 204 contiguous nucleic acids from SEQ ID NO:4 are also contemplated to be particularly useful.
The Endo-SR polypeptides of the present invention preferentially cleave either a xe2x80x9cTGGGNxe2x80x9d or a xe2x80x9cTGAGCxe2x80x9d polynucleotide sequence, and more preferably cleave both of these sequences, which are known as xe2x80x9cswitch pentamer motifsxe2x80x9d at or near recombination breakpoints. These polypeptides have been shown to be enriched in lymphoid tissue nuclear extracts.
In one important embodiment, the invention provides an isolated and purified amino acid segment comprising Endo-SR polypeptide comprising the amino acid sequence of SEQ ID NO:2. This polypeptide is a murine polypeptide, the coding region for which is given in SEQ ID NO:1 (a cDNA clone). The corresponding murine genomic DNA sequences are given in SEQ ID NO:3 and SEQ ID NO:4. The Endo-SR polypeptide exhibits specific endonuclease activity for G-rich DNA sequences. In related embodiments, methods for making and using this protein, derivatives and mutants thereof, and antibodies directed against these proteins are also disclosed. Also disclosed are methods for the design of inhibitors, such as peptidomimetics, of Endo-SR, and their use in modulation of Endo-SR activity.
In another important embodiment, the invention provides an isolated and purified nucleic acid segment comprising the mammalian gene which encodes the Endo-SR polypeptide disclosed herein. The nucleotide sequence of the cDNA is given in SEQ ID NO:1, and the partial murine genomic sequences are identified in SEQ ID NO:3 and SEQ ID NO:4. In related embodiments, methods for making, using, altering, mutagenizing, assaying, and quantitating these nucleic acid segments are also disclosed. Also disclosed are diagnostic methods and assay kits for the identification and detection of related gene sequences in a variety of in vitro and in vivo methodologies. Because Endo-SR has been implicated in the process of antibody isotype switch recombination and programmed cell death, highly purified or recombinant Endo-SR represents a useful tool for dissection of the mechanism of switch recombination and/or apoptosis.
Another aspect of the present invention is a mammalian cell, and in particular, a bovine or human cell that produces the novel Endo-SR polypeptide disclosed herein. Exemplary mammalian cells that produce the polypeptide include human HeLa and 293T kidney cells as well as mouse L-cell and NIH3T3.
A further aspect of the present invention is a vector, such as a plasmid, cosmid, phage, virus, phagemid, bacterial artificial chromosome or yeast artificial chromosome, that contains a nucleic acid sequence comprising a whole or a portion of a gene encoding Endo-SR (cDNA, SEQ NO ID:1; genomic clones, SEQ ID NO:3 and SEQ ID NO:4). Also provided is a transformed host cell comprising a native or recombinant gene encoding Endo-SR, as well as a tissue culture, a cell culture, or an animal, fungal or bacterial cell culture or suspension or lysate of a host cell transformed with such a vector.
The invention also provides a pharmaceutical composition comprising an Endo-SR polypeptide or a gene encoding such a polypeptide.
Because moderate levels of enzymatically active Endo-SR have been detected in animal serum and the Endo-SR is secreted, the polypeptide may play a role in preventing DNA accumulation in blood and tissues. Monoclonal or polyclonal antibodies directed towards the Endo-SR protein and/or Endo-SR peptides may prove to have therapeutic potential for patients carrying Endo-SR mutations resulting in elevated secretion of this protein. Alternatively, anti-Endo-SR antibodies may prove useful in the detection and diagnosis of patients with elevated or repressed levels of this enzyme. Therefore, in one embodiment, there is provided a monoclonal antibody that binds immunologically to an Endo-SR polypeptide. The antibody preferably is a mammalian antibody, and is non-cross-reactive with other mammalian polypeptides. Alternatively, the antibody may bind specifically to either human, or non-human Endo-SR polypeptides, but not to both human and non-human Endo-SR polypeptides. The antibody may further comprise a detectable label, such as a fluorescent label, a chemiluminescent label, a radiolabel or an enzyme. Also encompassed are hybridoma cells and cell lines producing such antibodies.
In another embodiment, there is included a polyclonal antisera, antibodies of which bind immunologically to an Endo-SR polypeptide. The antisera may be derived from any animal, but preferably is from an animal other than a human. Preferred antigens for the preparation of such sera include an Endo-SR polypeptide from human, bovine, murine, or other mammalian origins. A preferred host for the polyclonal antisera is preferably rabbit, goat, or other such animal. In an illustrative embodiment, rabbit polyclonal antisera raised against a recombinant human Endo-SR protein fragment was generated and shown to specifically recognize the immunizing antigen. The immunogen was generated from a Endo-SR gene fragment representing exons 5-6 of the human coding sequence cloned into the pET15b(trademark) expression vector (Novagen), which was subsequently expressed and purified from bacteria.
It also is an objective of the present invention to provide methods for making and using the Endo-SR compositions disclosed herein, and also for using the genes which encode them.
For example, the invention provides a method of obtaining an Endo-SR polypeptide from a mammalian cell. Endo-SR/DNase II activity was purified to homogeneity or near homogeneity from both mammals and C. elegans nematodes in a procedure in which crude protein extracts are selectively fractionated by successive ammonium sulfate precipitation, isoelectric focusing, cation-exchange chromatography, and size exclusion chromatography (essentially as per Lyon and Aguilera, 1997). Nuclear extracts are used as the starting material in Endo-SR purification from mammalian cell lines and tissues, while purification of the nematode enzyme begins with whole cell extracts. Endo-SR activity has been detected in cytoplasmic and nuclear extracts of all assayed tissues and cell lines.
Nuclease assays have demonstrated that, among the tissues tested, spleen contains the most Endo-SR specific activity, followed by thymus and liver. Spleen tissue was used as the source material for purification of the bovine enzyme due to its preferential enrichment in this activity. Endo-SR activity has also been detected at variable levels in all human and murine cell lines assayed to date and therefore this activity may be isolated from such cell lines essentially as previously described (Lyon, et al., 1996).
In another embodiment, there is provided a method of producing the Endo-SR polypeptide from a host cell transformed with one or more DNA sequences encoding the polypeptide. Such preparation is typically referred to in the art as xe2x80x9clarge-scalexe2x80x9d or xe2x80x9crecombinantxe2x80x9d protein production. In an exemplary method, the polypeptides of the present invention may be produced by a method that generally comprises:
Large amounts of Endo-SR protein could most efficiently be generated from a cell line containing an integrated Endo-SR expression vector. Endo-SR activity has been overexpressed by transient and stable transfection of mammalian cell lines with expression vector constructs. Since prolonged Endo-SR overexpression reduces cell viability, stable transfectants have been produced using the Tet-Off(trademark) (Clontech) system. Recombinant genes under the control of this system are expressed only in the absence of inhibitory concentrations of tetracycline and recombinant protein expression can be modulated by varying the concentration of tetracycline in the culture media. Since the Endo-SR protein contains a leader sequence and has been shown to be secreted from the cell, it is possible that recombinant protein will be easily isolated from cell supernatants in serum-free media. Other human (such as HeLa and 293T kidney cells) and mouse (such as L-cell, NIH3T3, or other cell lines) may also be used to obtain cell types that permit optimal high-level production of this enzyme in culture.
In another embodiment, there is provided a method of cleaving a polynucleotide such as DNA. This method generally involves contacting a polynucleotide that comprises at least one xe2x80x9cTGGGNxe2x80x9d or one xe2x80x9cTGAGCxe2x80x9d sequence with an amount of an Endo-SR polypeptide composition effective to cleave such polynucleotide.
In a further embodiment, there is provided a method for killing a tumor comprising the step of contacting a tumor cell within a subject with a therapeutically-effective amount of an Endo-SR polypeptide compositions. Preferably, the tumor cell is within a mammal such as a human.
In still a further embodiment, there is provided a method for treating cancer comprising the step of contacting a tumor cell within a subject with a nucleic acid (a) encoding an Endo-SR polypeptide and (b) a promoter operable in the tumor cell, wherein the promoter is operatively linked to the region encoding the Endo-SR polypeptide, under conditions permitting the uptake of the nucleic acid by the tumor cell. The subject is preferably an animal, and most preferably a human.
In still yet a further embodiment, there is provided a transgenic mammal in which both genomic copies of the gene encoding an Endo-SR polypeptide are interrupted, deleted, or replaced with at least one other gene or gene fragment. Such an animal is typically referred to as an Endo-SR xe2x80x9cknockoutxe2x80x9d animal. Such animals are contemplated to be excellent models for gene therapy/DNA vaccines as they may allow DNA based vectors to efficiently integrate into the host genome. The mammal is preferably murine, porcine, ovine, epine, lupine, equine, bovine, caprine, canine, or feline.
In still yet an additional embodiment, there is provided a method of screening a candidate substance which inhibits Endo-SR activity in a cell. This method generally comprises (a) providing a cell having functional Endo-SR polypeptide activity; (b) contacting the cell with the candidate substance; and (c) determining the inhibitory effect of the candidate substance on the Endo-SR activity in the cell. The candidate substance may be a chemotherapeutic or radiotherapeutic agent or be selected from a small molecule library, or alternatively, a peptidomimetic which inhibits Endo-SR activity. Endo-SR inhibition may result in similar effect as the mutation of the gene in animals. The cell may be contacted in vitro or in vivo.
The foregoing objects of the invention and others that are now readily apparent to those of skill in the art having the benefit of the present disclosure are described more fully in the sections which follow:
2.1 Polynucleotide Compositions
The present invention also concerns polynucleotide segments, (including DNA, RNA, and PNAs) that can be isolated from virtually any source, that are free from total genomic DNA and that encode the whole or a portion of the novel endonuclease disclosed herein. The gene having the nucleotide sequence of SEQ ID NO:1 encodes the murine Endo-SR polypeptide having an amino acid sequence shown in SEQ ID NO:2. DNA segments encoding this polypeptide may be used to identify additional DNA sequences, that may encode proteins, polypeptides, subunits, functional domains, and the like of Endo-SR-related or other non-related gene products. In addition these DNA segments may be synthesized entirely in vitro using methods that are well-known to those of skill in the art.
As used herein, the term xe2x80x9cDNA segmentxe2x80x9d refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding an Endo-SR polypeptide refers to a DNA segment that contains an Endo-SR polypeptide coding sequence yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term xe2x80x9cDNA segmentxe2x80x9d, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, BACs, YACs, viruses, and the like. In one embodiment, the inventors have constructed BAC genomic clones which find important utility in the generation of xe2x80x9cknockoutxe2x80x9d animals.
Similarly, a DNA segment comprising an isolated or purified Endo-SR polypeptide-encoding gene refers to a DNA segment which may include in addition to peptide encoding sequences, certain other elements such as, regulatory sequences, isolated substantially away from other naturally occurring genes or protein-encoding sequences. In this respect, the term xe2x80x9cgenexe2x80x9d is used for simplicity to refer to a functional protein-, polypeptide- or peptide-encoding unit. As will be understood by those in the art, this functional term includes not only genomic sequences, including extrachromosomal DNA sequences, but also promoter or cis acting sequences and/or engineered gene segments that express, or may be adapted to express one or more proteins, polypeptides or peptide fragments.
xe2x80x9cIsolated substantially away from other coding sequencesxe2x80x9d means that the gene of interest, in this case, an Endo-SR polypeptide-encoding gene, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or operon coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes, recombinant genes, synthetic linkers, or coding regions later added to the segment by the hand of man.
In particular embodiments, the invention concerns isolated polynucleotides and recombinant vectors incorporating polynucleotide sequences that encode all or part of an Endo-SR polypeptide species that includes within its amino acid sequence an at least 19 contiguous amino acid sequence from SEQ ID NO:2. More preferably, the polynucleotide comprises a nucleic acid sequence that encodes an Endo-SR polypeptide species that includes within its amino acid sequence an at least twenty amino acid contiguous sequence of SEQ ID NO:2. Still more preferably, the polynucleotide comprises a nucleic acid sequence that encodes an Endo-SR polypeptide species that includes within its amino acid sequence an at least 22 amino acid contiguous sequence of SEQ ID NO:2. In illustrative embodiments, such a polynucleotide comprises at least about 24, at least about 26, at least about 28, or at least about 30 or more contiguous amino acids from SEQ ID NO:2. In one embodiment, the preferred polynucleotides of the invention encode a polypeptide that comprises the sequence of SEQ ID NO:2.
The term xe2x80x9ca sequence essentially as set forth in SEQ ID NO:2,xe2x80x9d means that the sequence substantially corresponds to a portion of the sequence of SEQ ID NO:2 and has relatively few amino acids that are not identical to, or a biologically functional equivalent of, the amino acids of any of these sequences. The term xe2x80x9cbiologically functional equivalentxe2x80x9d is well understood in the art and is further defined in detail herein (e.g., see Illustrative Embodiments). Accordingly, sequences that have between about 70% and about 80%, or more preferably between about 81% and about 90%, or even more preferably between about 91% and about 99% amino acid sequence identity or functional equivalence to the amino acids of SEQ ID NO:2 will be sequences that are xe2x80x9cessentially as set forth in SEQ ID NO:2.xe2x80x9d In illustrative embodiments, the endonuclease polypeptides of the present invention emcompass polypeptide species that are about 92% identical to the amino acid sequence of SEQ ID NO:2, or about 93% identical to the amino acid sequence of SEQ ID NO:2, or even about 94% identical to the amino acid sequence of SEQ ID NO:2, and in some instances will be about 95% or 96% identical to the amino acid sequence of SEQ ID NO:2, or even more. As such, it is contemplated that sequences that are about 97% or 98% or 99% identical to the amino acid sequence of SEQ ID NO:2 will be preferred for practice of the present invention. Indeed, when mutant endonuclease compositions are contemplated, or when endonuclease polypeptides are isolated from related species, or when allelic variants of the polypeptide species occur within one or more species of animal from which the polypeptide is isolated, it is contemplated that one or more amino acids may be altered, mutated, deleted, or even one or more amino acids added to the sequence of SEQ ID NO:2, and still have a polypeptide that has endonuclease activity. Such mutated polypeptide species may have altered endonuclease activity or altered endonuclease specificity, as as such, may possess greater or reduced activity or specificity when compared to the wild-type polypeptide shown in SEQ ID NO:2.
It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5xe2x80x2 or 3xe2x80x2 sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5xe2x80x2 or 3xe2x80x2 portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other polynucleotide sequences, such as promoters, enhancers, expression elements, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant polynucleotide protocol. For example, nucleic acid fragments may be prepared that include a short contiguous stretch encoding the whole or a portion of the peptide sequence disclosed in SEQ ID NO:2, or that are identical to or complementary to DNA sequences which encode the peptide disclosed in SEQ ID NO:2, and particularly the DNA segment disclosed in SEQ ID NO:1. For example, DNA sequences such as about 24, about 25, about 26, or about 27 or more nucleotides in length, and even those that are up to and including about 10,000, about 5,000, about 4,000, about 3,000, about 2,000, about 1000, about 800, about 700, about 600, about 500, about 400, about 300, about 200, about 100, or even about 75, 50 or 25 base pairs in length (including all intermediate lengths) are also contemplated to be useful.
It will be understood that this invention is not limited to the particular nucleic acid sequences which encode peptides of the present invention, or which encode the amino acid sequence of SEQ ID NO:2, including the DNA sequence which is particularly disclosed in SEQ ID NO:1, and the genomic DNA sequences disclosed in SEQ ID NO:3 and SEQ ID NO:4. Recombinant vectors and isolated DNA segments may therefore variously include the peptide-coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include these peptide-coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
It will be readily understood that xe2x80x9cintermediate lengthsxe2x80x9d, in the contexts of both polynucleotides and polypeptides, means any integer length that lies within or is between the quoted ranges. For example, sequences from between about 24 and about 10,000 nucleotides in length, will include all integers between such lengths, including lengths of about 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49, etc.; 50, 51, 52, etc.; 60, 61, 62, etc., 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 105, 110, 115, etc.; 120, 130, 140, 150, 160, 170, 180, 190, etc.; 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, including all integers through the 301-500; 501-1,000; 1,001-2,000; 2,001-3,000; 3,001-4,000; 4,001-6,000, 6,001-9,000, and up to and including sequences of about 9,500, 9,600, 9,700, 9,800, 9,900, 10,000 nucleotides and the like. Similarly, sequences from between about 19 and about 200 amino acids in length, will include all integers between such lengths, including lengths of about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, etc.; 40, 41, 42, 43, etc.; 50, 51, 52, etc.; 60, 61, 62, etc., 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, etc.; 120, 130, 140, 150, 160, 170, 180, 190, etc.; including all integers up to and including the full-length polypeptide of 192 amino acids. Naturally, when all or a portion of the coding region for the Endo-SR polypeptide is fused to one or more additional polypeptides (such as in the preparation of fusion proteins, peptide adjuvants, and the like) polypeptides longer than the full-length sequence are also contemplated to be useful. Likewise, the Endo-SR polypeptide homologs isolated from other mammalian species may be slightly longer than the sequence of SEQ ID NO:2, and as such, polypeptides having a length of about 192, about 194, about 195, about 196, about 197, about 198, about 199, or even about 200 to about 300; or more amino acids and the like may also be useful in certain embodiments, and as such, are contemplated to fall within the scope of the present disclosure.
The polynucleotides and polypeptides of the present invention also encompass biologically-functional, equivalent polypeptides and polynucleotides. Such sequences, for example, may arise as a consequence of codon redundancy and functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Because, it has been demonstrated that the Endo-SR exhibits both specific endonuclease activity (at G-rich residues) and a degradative activity, it is likely that the xe2x80x9cimprovedxe2x80x9d or xe2x80x9csecond-generationxe2x80x9d Endo-SR polypeptides may be desirable and synthesized to improve or modify its sequence specificity. Such altered proteins may result in novel endonuclease activities with specificity resembling that of prokaryotic restriction endonucleases with many potential application in the research and biotechnology fields. As such, functionally-equivalent Endo-SR proteins or peptides also represent important aspects of the invention, and may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the antigenicity of the protein or to test mutants in order to examine activity at the molecular level.
If desired, one may also prepare fusion proteins and peptides, e.g., where the peptide-coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e.g., proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
Recombinant vectors form further aspects of the present invention. Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length Endo-SR protein or smaller peptide fragment derived therefrom, is positioned under the control of one or more promoters or other expression elements. The promoter may be in the form of the promoter that is naturally associated with a gene encoding peptides of the present invention, as may be obtained by isolating the 5xe2x80x2 non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or PCR(trademark) technology, in connection with the compositions disclosed herein.
2.2 Polynucleotides as Hybridization Probes and Primers
In addition to their use in directing the expression of the novel Endo-SR polypeptides of the present invention, the nucleic acid sequences contemplated herein also have a variety of other uses. For example, they also have utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that nucleic acid segments that comprise a sequence region that consists of at least a 14 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 14 nucleotide long contiguous DNA segment of SEQ ID NO:1 will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000, 2000, 5000, 10000, etc. (including all intermediate lengths and up to and including full-length sequences) will also be of use in certain embodiments.
The ability of such nucleic acid probes to specifically hybridize to an Endo-SR polypeptide-encoding sequence will enable them to be of use in detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having sequence regions consisting of contiguous nucleotide stretches of 14-25, 26-40, 41-60, or even of 100-200 nucleotides or so in length, either identical to, or complementary to, the sequence disclosed in SEQ ID NO:1, are particularly contemplated as hybridization probes for use in, e.g., Southern and Northern blotting. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the contiguous complementary region may be varied, such as between about 10-14 and about 100 or 200 nucleotides, but larger contiguous complementarity stretches may be used, according to the length complementary sequences one wishes to detect.
The use of a hybridization probe of about 19 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 19 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 contiguous nucleotides, or even longer where desired.
Of course, fragments may also be obtained by other techniques such as, e.g., by mechanical shearing or by restriction enzyme digestion. Small nucleic acid segments or fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Also, fragments may be obtained by application of nucleic acid reproduction technology, such as the PCR(trademark) technology of U.S. Pat. No. 4,683,195 and U.S. Pat. No. 4,683,202 (each specifically incorporated herein by reference in its entirety), by introducing selected sequences into recombinant vectors for recombinant production, and by other recombinant DNA techniques generally known to those of skill in the art of molecular biology.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNA fragments. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50xc2x0 C. to about 70xc2x0 C. Such selective conditions, typically referred to in the art as xe2x80x9chigh stringency conditionsxe2x80x9d tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating Endo-SR-encoding DNA segments. Detection of DNA segments via hybridization is well-known to those of skill in the art, and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporated herein by reference) are exemplary of the methods of hybridization analyses. Teachings such as those found in the texts of Maloy et al., 1994; Segal 1976; Prokop, 1991; and Kuby, 1991, are particularly relevant.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate Endo-SR-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as salt concentrations of from about 0.15 M to about 0.9 M, and temperature ranges of from about 20xc2x0 C. to about 55xc2x0 C. Using these conditions, typically referred to in the art as xe2x80x9clow stringency conditions,xe2x80x9d cross-hybridizing species are readily identified as positively hybridizing signals with respect to control hybridizations.
In any case, however, it is generally appreciated that hybridization conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results. In any circumstances, it is desirable that the conditions used for hybridization to detect gene segments encoding Endo-SR polypeptides will be sufficiently stringent to permit hybridization of the probe to sequences which encode polypeptides having Endo-SR or Endo-SR-like activity, but will not permit hybridization of the probe to nucleotide sequences which do not encode polypeptides having Endo-SR or Endo-SR-like activity.
In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantitated, by means of the label.
2.3 Recombinant Vectors and Protein Expression
The invention also discloses and claims a composition comprising an Endo-SR polypeptide. The composition may comprises one or more host cells which express an Endo-SR polypeptide, recombinant host cells expresses the protein, cell suspensions, extracts, inclusion bodies, or tissue cultures or culture extracts which contain an Endo-SR polypeptide, culture supernatant, disrupted cells, cell extracts, lysates, homogenates, and the like. The Endo-SR polypeptides may be present in aqueous form, or alternatively, in dry, semi-wet, or similar forms such as cell paste, cell pellets, or alternatively freeze dried, powdered, lyophilized, evaporated, or otherwise similarly prepared in dry form. Such means for preparing Endo-SR polypeptides are well-known to those of skill in the art of protein isolation and purification. In certain embodiments, the Endo-SR polypeptides may be purified, concentrated, admixed with other reagents, or processed to a desired final form. Preferably, the composition will comprise from about 1% to about 90% by weight of the Endo-SR polypeptide, and more preferably from about 5% to about 50% by weight.
In a preferred embodiment, the Endo-SR polypeptide compositions of the invention may be prepared by a process which comprises the steps of culturing a host cell which expresses an Endo-SR polypeptide under conditions effective to produce such a protein, and then obtaining the protein from the cell. The obtaining of such an Endo-SR polypeptide may further include purifying, concentrating, processing, or admixing the protein with one or more reagents. Preferably, the Endo-SR polypeptide is obtained in an amount of from between about 1% to about 90% by weight, and more preferably from about 5% to about 70% by weight, and even more preferably from about 10% to about 20% to about 30%, or even to about 40% or 50% by weight.
The invention also relates to a method of preparing an Endo-SR polypeptide composition. Such a method generally involves the steps of culturing a host cell which expresses an Endo-SR polypeptide under conditions effective to produce the protein, and then obtaining the protein so produced. The vectors of the invention which comprise nucleic acid segments encoding Endo-SR polypeptides may be used to transform other suitable bacterial or eukaryotic cells to produce the polypeptides of the invention.
In such embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a DNA segment encoding an Endo-SR polypeptide in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, or eukaryotic cell. Preferred eukaryotic cells are animal cells, with mammalian cells, particularly human cells, being most preferred. Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type, tissue, organism, animal, or recombinant host cell chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al., 1989. The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides. Appropriate promoter systems contemplated for use in high-level expression include, but are not limited to, the Pichia expression vector system (Pharmacia LKB Biotechnology).
In connection with expression embodiments to prepare recombinant proteins and peptides, it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire peptide sequence being most preferred. However, it will be appreciated that the use of shorter DNA segments to direct the expression of Endo-SR polypeptides or epitopic core regions, such as may be used to generate anti-Endo-SR antibodies, also falls within the scope of the invention. DNA segments that encode peptide antigens from about 8 to about 50 amino acids in length, or more preferably, from about 8 to about 30 amino acids in length, or even more preferably, from about 8 to about 20 amino acids in length are contemplated to be particularly useful. Such peptide epitopes may be amino acid sequences which comprise contiguous amino acid sequences from SEQ ID NO:2.
2.4 Transgenes and Transgenic Host Cells Expressing Endo-SR
In yet another aspect, the present invention provides methods for producing a transgenic cell, and in particular a plant or animal cell which expresses a nucleic acid segment encoding the novel Endo-SR polypeptides of the present invention. The process of producing transgenic cells is well-known in the art. In general, the method comprises transforming a suitable host cell with a DNA segment which contains a promoter operably linked to a coding region that encodes an Endo-SR polypeptide. Such a coding region is generally operatively linked to a transcription-terminating region, whereby the promoter is capable of driving the transcription of the coding region in the cell, and hence providing the cell the ability to produce the recombinant protein in vivo. Alternatively, in instances where it is desirable to control, regulate, or decrease the amount of a particular Endo-SR polypeptide expressed in a particular transgenic cell, the invention also provides for the expression of Endo-SR antisense mRNA. The use of antisense mRNA as a means of controlling or decreasing the amount of a given protein of interest in a cell is well-known in the art.
In a preferred embodiment, the invention encompasses an animal cell which has been transformed with a nucleic acid segment of the invention, and which expresses a gene or gene segment encoding one or more of the novel polypeptide compositions disclosed herein. As used herein, the term xe2x80x9ctransgenic host cellxe2x80x9d is intended to refer to a host cell, either prokaryotic or eukaryotic, that has incorporated DNA sequences, including but not limited to genes which are perhaps not normally present, DNA sequences not normally transcribed into RNA or translated into a protein (xe2x80x9cexpressedxe2x80x9d), or any other genes or DNA sequences which one desires to introduce into the non-transformed host cell, such as genes which may normally be present in the non-transformed cell but which one desires to either genetically engineer or to have altered expression.
It is contemplated that in some instances the genome of a transgenic host cell of the present invention will have been augmented through the stable introduction of a transgene encoding an Endo-SR polypeptide, either native or synthetically modified or mutated. In some instances, more than one transgene will be incorporated into the genome of the transformed host cell. Such is the case when more than one Endo-SR polypeptide-encoding DNA segment is incorporated into the genome of such a cell. In certain situations, it may be desirable to have one, two, three, four, or even more Endo-SR polypeptides (either native or recombinantly-engineered) incorporated and stably expressed in the transformed transgenic host cell. In preferred embodiments, the introduction of the transgene into the genome of the host cell results in a stable integration wherein the progeny of such cells also contain a copy of the transgene in their genome.
2.5 Transgenic Animals Lacking Endo-SR Activity
Alternatively, the invention provides a xe2x80x9cknockoutxe2x80x9d transgenic animal which has been transformed with a nucleic acid segment of the invention, in such a way that Endo-SR activity is reduced or eliminated from cells of the transgenic knockout animal. In some instances the genome of a transgenic host cell of the present invention will have been augmented through the stable replacement of a native Endo-SR encoding gene with a defective Endo-SR gene, or alternatively, the native Endo-SR-encoding gene will be disrupted, deleted, or replaced by a nucleic acid segment which prevents synthesis of a functional Endo-SR polypeptide in the resulting transgenic animal.
Means for transforming a host cell and the preparation of a transgenic cell line are well-known in the art (as exemplified in U.S. Pat. Nos. 5,550,318; 5,508,468; 5,482,852; 5,384,253; 5,276,269; and 5,225,341, all specifically incorporated herein by reference), and are briefly discussed herein. Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segments for use in transforming such cells will, of course, generally comprise either the operons, genes, or gene-derived sequences of the present invention, either native, or synthetically-derived, and particularly those encoding the disclosed Endo-SR polypeptides. These DNA constructs can further include structures such as promoters, enhancers, polylinkers, or even gene sequences which have positively- or negatively-regulating activity upon the particular genes of interest as desired. The DNA segment or gene may encode either a native or modified Endo-SR polypeptide, which will be expressed in the resultant recombinant cells, and/or which will impart a desired phenotype to the transformed host cell.
2.6 Site-Specific Mutagenesis
Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications. As will be appreciated, the technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage. These phage are readily commercially available and their use is generally well known to those skilled in the art. Double stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
The preparation of sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained. For example, recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
2.7 Compositions and Methods for Producing Antibodies
In particular embodiments, the inventors contemplate the use of antibodies, either monoclonal or polyclonal which bind to the Endo-SR polypeptides disclosed herein. Means for preparing and characterizing antibodies are well known in the art (See, e.g, Harlow and Lane, 1988; incorporated herein by reference). The methods for generating monoclonal antibodies (mAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund""s adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund""s adjuvants and aluminum hydroxide adjuvant.
The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified Endo-SR polypeptide. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5xc3x97107 to 2xc3x97108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
One preferred murine myeloma cell is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986, pp. 71-74).
Fusion procedures usually produce viable hybrids at low frequencies, about 1xc3x9710xe2x88x926 to 1xc3x9710xe2x88x928. However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.
The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration. The individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
2.8 Endo-SR Screening and Immunodetection Kits
The present invention also provides compositions, methods and kits for screening samples suspected of containing an Endo-SR polypeptide or a gene encoding such an Endo-SR polypeptide. Alternatively, the invention provides compositions, methods and kits for screening samples suspected of containing Endo-SR polypeptides or genes encoding Endo-SR polypeptides which are functionally equivalent to, or substantially homologous to, the Endo-SR polypeptide disclosed herein. Such screening may be performed on samples such as transformed host cells, clinical or laboratory samples suspected of containing or producing such a polypeptide or nucleic acid segment. A kit can contain a novel nucleic acid segment or an antibody of the present invention. The kit can contain reagents for detecting an interaction between a sample and a nucleic acid or an antibody of the present invention. The provided reagent can be radio-, fluorescently- or enzymatically-labeled. The kit can contain a known radiolabeled agent capable of binding or interacting with a nucleic acid or antibody of the present invention.
The reagent of the kit can be provided as a liquid solution, attached to a solid support or as a dried powder. Preferably, when the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. Preferably, when the reagent provided is attached to a solid support, the solid support can be chromatograph media, a test plate having a plurality of wells, or a microscope slide. When the reagent provided is a dry powder, the powder can be reconstituted by the addition of a suitable solvent, that may be provided.
In still further embodiments, the present invention concerns immunodetection methods and associated kits. It is proposed that the Endo-SR polypeptides of the present invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect Endo-SR polypeptides or Endo-SR polypeptide-related epitope-containing containing amino acid sequences. In general, these methods will include first obtaining a sample suspected of containing such a protein, peptide or antibody, contacting the sample with an antibody or peptide in accordance with the present invention, as the case may be, under conditions effective to allow the formation of an immunocomplex, and then detecting the presence of the immunocomplex.
In general, the detection of immunocomplex formation is quite well known in the art and may be achieved through the application of numerous approaches. For example, the present invention contemplates the application of ELISA, RIA, immunoblot (e.g., dot blot), indirect immunofluorescence techniques and the like. Generally, immunocomplex formation will be detected through the use of a label, such as a radiolabel or an enzyme tag (such as alkaline phosphatase, horseradish peroxidase, or the like). Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody or a biotin/avidin ligand binding arrangement, as is known in the art.
For assaying purposes, it is proposed that virtually any sample suspected of comprising either an Endo-SR polypeptide or an Endo-SR polypeptide-related peptide or antibody sought to be detected, as the case may be, may be employed. It is contemplated that such embodiments may have application in the titering of antigen or antibody samples, in the selection of hybridomas, and the like. In related embodiments, the present invention contemplates the preparation of kits that may be employed to detect the presence of Endo-SR polypeptides or related peptides and/or antibodies in a sample. Samples may include cells, cell supernatants, cell suspensions, cell extracts, enzyme fractions, protein extracts, or other cell-free compositions suspected of containing Endo-SR polypeptides. Generally speaking, kits in accordance with the present invention will include a suitable Endo-SR polypeptide, peptide or an antibody directed against such a protein or peptide, together with an immunodetection reagent and a means for containing the antibody or antigen and reagent. The immunodetection reagent will typically comprise a label associated with the antibody or antigen, or associated with a secondary binding ligand. Exemplary ligands might include a secondary antibody directed against the first antibody or antigen or a biotin or avidin (or streptavidin) ligand having an associated label. Of course, as noted above, a number of exemplary labels are known in the art and all such labels may be employed in connection with the present invention.
The container will generally include a vial into which the antibody, antigen or detection reagent may be placed, and preferably suitably aliquotted. The kits of the present invention will also typically include a means for containing the antibody, antigen, and reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
2.9 ELISAs and Immunoprecipitation
ELISAs may be used in conjunction with the invention. In an ELISA assay, proteins or peptides incorporating Endo-SR polypeptide antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of milk powder. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
After binding of antigenic material to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween(copyright). These added agents also tend to assist in the reduction of nonspecific background. The layered antisera is then allowed to incubate for from about 2 to about 4 hours, at temperatures preferably on the order of about 25xc2x0 to about 27xc2x0 C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween(copyright), or borate buffer.
Following formation of specific immunocomplexes between the test sample and the bound antigen, and subsequent washing, the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for the first. To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the antisera-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g, incubation for 2 hours at room temperature in a PBS-containing solution such as PBS Tween(copyright)).
After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2xe2x80x2-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H2O2, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
The anti-Endo-SR antibodies of the present invention are particularly useful for the isolation of other Endo-SR polypeptide antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g. enzyme-substrate pairs.
2.10 Western Blots
The Endo-SR compositions of the present invention will find great use in immunoblot or western blot analysis. The anti-Endo-SR antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof. In conjunction with immuno-precipitation, followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background. This is especially useful when the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
2.11 Epitopic Core Sequences
The present invention is also directed to Endo-SR polypeptide compositions, free from total cells and other peptides, which comprise a purified Endo-SR polypeptide which incorporates an epitope that is immunologically cross-reactive with one or more anti-Endo-SR antibodies. In particular, the invention concerns epitopic core sequences derived from Endo-SR polypeptides and Endo-SR polypeptide-derived proteins or peptides.
As used herein, the term xe2x80x9cincorporating an epitope(s) that is immunologically cross-reactive with one or more anti-Endo-SR antibodiesxe2x80x9d is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within an Endo-SR polypeptide. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the Endo-SR polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art.
The identification of Endo-SR immunodominant epitopes, and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter. For example, one may employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, e.g., Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these xe2x80x9cepitopic core sequencesxe2x80x9d may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
Preferred peptides for use in accordance with the present invention will generally be on the order of about 8 to about 20 amino acids in length, and more preferably about 8 to about 15 amino acids in length. It is proposed that shorter antigenic peptides will provide advantages in certain circumstances, for example, in the preparation of immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
It is proposed that particular advantages of the present invention may be realized through the preparation of synthetic peptides which include modified and/or extended epitopic/immunogenic core sequences which result in a xe2x80x9cuniversalxe2x80x9d epitopic peptide directed to Endo-SR polypeptides. These epitopic core sequences are identified herein in particular aspects as hydrophilic regions of the particular polypeptide antigen. It is proposed that these regions represent those which are most likely to promote T-cell or B-cell stimulation, and, hence, elicit specific antibody production.
An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is xe2x80x9ccomplementaryxe2x80x9d to, and therefore will bind, antigen binding sites on the Endo-SR polypeptide-directed antibodies disclosed herein. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term xe2x80x9ccomplementaryxe2x80x9d refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence anticipated by the present disclosure would generally be on the order of about 8 amino acids in length, with sequences on the order of 10 to 20 being more preferred. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention. However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
The identification of epitopic core sequences is known to those of skill in the art, for example, as described in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. Moreover, numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysis programs (e.g., DNAStar(copyright) software, DNAStar, Inc., Madison, Wis.) may also be useful in designing synthetic peptides in accordance with the present disclosure.
Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence, are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of commercially available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquotted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at about 4xc2x0 C., or more preferably, frozen. Of course, where the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.
2.12 Biological Functional Equivalents
Modification and changes may be made in the structure of the Endo-SR polypeptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the codons given in Table 1.
For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein""s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (xe2x88x920.4); threonine (xe2x88x920.7); serine (xe2x88x920.8); tryptophan (xe2x88x920.9); tyrosine (xe2x88x921.3); proline (xe2x88x921.6); histidine (xe2x88x923.2); glutamate (xe2x88x923.5); glutamine (xe2x88x923.5); aspartate (xe2x88x923.5); asparagine (xe2x88x923.5); lysine (xe2x88x923.9); and arginine (xe2x88x924.5).
It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, ie., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within xc2x12 is preferred, those which are within xc2x11 are particularly preferred, and those within xc2x10.5 are even more particularly preferred.
It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.
As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0xc2x11); glutamate (+3.0xc2x11); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (xe2x88x920.4); proline (xe2x88x920.5xc2x11); alanine (xe2x88x920.5); histidine (xe2x88x920.5); cysteine (xe2x88x921.0); methionine (xe2x88x921.3); valine (xe2x88x921.5); leucine (xe2x88x921.8); isoleucine (xe2x88x921.8); tyrosine (xe2x88x922.3); phenylalanine (xe2x88x922.5); tryptophan (xe2x88x923.4).
It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids
whose hydrophilicity values are within xc2x12 is preferred, those which are within +1 are particularly preferred, and those within xc2x10.5 are even more particularly preferred.
As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
2.13 Regulating the Expression of Endo-SR Activity in a Cell Using Anti-Sense Constructs
The end result of the flow of genetic information is the synthesis of protein. DNA is transcribed by polymerases into messenger RNA and translated on the ribosome to yield a folded, functional protein. Thus, even from this simplistic description of an extremely complex set of reactions, it is obvious that there are several steps along the route where protein synthesis can be inhibited. The native DNA segment coding for Endo-SR, as all such mammalian DNA strands, has two strands: a sense strand and an antisense strand held together by hydrogen bonding. The messenger RNA coding for Endo-SR has the same nucleotide sequence as the sense DNA strand except that the DNA thymidine is replaced by uridine. Thus, synthetic antisense nucleotide sequences will bind to the mRNA coding for Endo-SR and inhibit expression of the protein.
Antisense oligodeoxynucleotides (AS-ODNs) are single-stranded, short sequences of DNA (Cohen, 1989; De Mesmaeker et al., 1995) that are complementary to specific messenger RNA (mRNA). Since AS-ODNs hybridize with the mRNA, they prevent the targeted mRNA from expressing its polypeptide product in the cell.
The targeting of antisense oligonucleotides to bind mRNA is one mechanism to shut down protein synthesis. For example, the synthesis of polygalactauronase and the muscarine type 2 acetylcholine receptor are inhibited by antisense oligonucleotides directed to their respective mRNA sequences (U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829, each specifically incorporated herein by reference in its entirety). Further, examples of antisense inhibition have been demonstrated with the nuclear protein cyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin, STK-1, striatal GABAA receptor and human EGF (Jaskulski et al., 1988; Vasanthakumar and Ahmed, 1989; Peris et al., 1998; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573; U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288, each specifically incorporated herein by reference in its entirety). Antisense constructs have also been described that inhibit and can be used to treat a variety of abnormal cellular proliferations, e.g cancer (U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No. 5,783,683, each specifically incorporated herein by reference in its entirety).
AS-ODNs have also been used for the treatment of a variety of diseases and disorders in animals, including hypertension (Phillips, 1997; Wielbo et al., 1994; Phillips et al., 1994 and Gyurko et al., 1997; Gyurko et al., 1993; Meng et al., 1994; Wielbo et al., 1997; Wielbo et al., 1996; Wielbo et al., 1995).
The inventors contemplate that antisense oligonucleotide and peptide nucleic acid compositions that specifically bind Endo-SR mRNA in a mammalian cell, may be used to alter the expression of Endo-SR in the cell.
Therefore, the present invention provides a composition comprising at least a first oligonucleotide of at least 9 to about 35 bases in length, wherein the oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding a mammalian Endo-SR polypeptide, and further wherein binding of the oligonucleotide to the mRNA is effective in decreasing the activity of the enzyme in a host cell expressing the mRNA.
In certain aspects of the invention, the oligonucleotide comprises deoxyribonucleic acid, ribonucleic acid, or peptide-nucleic acid. In particular embodiments, the oligonucleotide comprises a sequence that is complementary to at least ten, at least eleven, at least twelve, at least thirteen or at least fourteen or more contiguous bases from SEQ ID NO:1. In other aspects of the present invention, the oligonucleotide has no more than 4, no more than 3, no more than 2, no more than 1 or no mismatches from the mRNA sequence to which it specifically binds. In particular aspects of the invention, the composition further comprises at least a second oligonucleotide of at least 9 to about 35 nucleotides in length, wherein the second oligonucleotide specifically binds to a portion of mRNA expressed from a gene encoding another mammalian endonuclease. Alternatively, the composition may further comprises one or more anti-cancer or anti-tumor agents, or may comprise one or more additional endonuclease compositions. In certain preferred embodiments, the composition further comprises a pharmaceutically-acceptible vehicle, exemplified by, but not limited to, a liposome, a lipid particle, a lipid vesicle, a nanoparticle, a microparticle, a nanocapsule, a nanosphere, or a sphingosome.
In certain aspects of the invention, the enzyme is a human enzyme. In particular embodiments, the host cell is a mammalian host cell. In certain preferred embodiments of the invention, the host cell is a human cell. In other preferred aspects, the host cell is comprised within a human.
The present invention also provides a polynucleotide of at least 9 to about 35 bases in length, wherein the polynucleotide specifically binds to a portion of mRNA expressed from a DNA segment encoding a mammalian Endo-SR polypeptide, and further wherein binding of the polynucleotide to the mRNA is effective in decreasing the transcription of the mRNA in a host cell expressing the mRNA. The present invention further provides an antisense nucleic acid molecule comprising a segment complementary to a sequence unique to mammalian Endo-SR-specific mRNA, wherein when administered to a living organism, the antisense molecule is capable of reducing the amount of the enzyme in the organism.
2.14 Definitions
The oligonucleotides (or xe2x80x9cODNsxe2x80x9d or xe2x80x9cpolynucleotidesxe2x80x9d or xe2x80x9coligosxe2x80x9d or xe2x80x9coligomersxe2x80x9d or xe2x80x9cn-mersxe2x80x9d) of the present invention are preferably deoxyoligonucleotides (i.e. DNAs), or derivatives thereof; ribo-oligonucleotides (i.e. RNAs) or derivatives thereof; or peptide nucleic acids (PNAs) or derivatives thereof.
The term xe2x80x9csubstantially complementary,xe2x80x9d when used to define either amino acid or nucleic acid sequences, means that a particular subject sequence, for example, an oligonucleotide sequence, is substantially complementary to the sequence of Endo-SR-specific sequences (e.g., those identified in SEQ ID NO:1 or SEQ ID NO:2), and thus will specifically bind to a portion of an mRNA encoding an Endo-SR polypeptide. As such, typically the sequences will be highly complementary to the mRNA xe2x80x9ctargetxe2x80x9d sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence. In many instances, it may be desirable for the sequences to be exact matches, i.e. be completely complementary to the sequence to which the oligonucleotide specifically binds, and therefore have zero mismatches along the complementary stretch. As such, highly complementary sequences will typically bind quite specifically to the target sequence region of the mRNA and will therefore be highly efficient in reducing, and/or even inhibiting the translation of the target mRNA sequence into polypeptide product.
Substantially complementary oligonucleotide sequences will be greater than about 80 percent complementary (or xe2x80x98% exact-matchxe2x80x99) to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and will, more preferably be greater than about 85 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds. In certain aspects, as described above, it will be desirable to have even more substantially complementary oligonucleotide sequences for use in the practice of the invention, and in such instances, the oligonucleotide sequences will be greater than about 90 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and may in certain embodiments be greater than about 95 percent complementary to the corresponding mRNA target sequence to which the oligonucleotide specifically binds, and even up to and including 96%, 97%, 98%, 99%, and even 100% exact match complementary to the target mRNA to which the designed oligonucleotide specifically binds.
Percent similarity or percent complementary of any of the disclosed sequences may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
xe2x80x9cRecombinant,xe2x80x9d as used herein, means that a protein is derived from recombinant (e.g., microbial or mammalian) expression systems. xe2x80x9cMicrobialxe2x80x9d refers to recombinant proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, xe2x80x9crecombinant microbialxe2x80x9d defines a protein produced in a microbial expression system which is essentially free of native endogenous substances. Protein expressed in most bacterial cultures, e.g., E. coli, will be free of glycan. Protein expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.
xe2x80x9cBiologically active,xe2x80x9d as used throughout the specification means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of specifically cross-react or bind specifically to one or more antibodies raised against a mammalian Endo-SR polypeptide or peptide fragment thereof.
xe2x80x9cDNA sequencexe2x80x9d refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct. Preferably, the DNA sequences are in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5xe2x80x2 or 3xe2x80x2 from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
xe2x80x9cRNA sequencexe2x80x9d refers to an RNA polymer, in the form of a separate fragment or as a component of a larger RNA construct, such as a messenger RNA (mRNA) encoding one or more Endo-SR polypeptides. Preferably, the RNA sequences are in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector, or alternatively, by chemically synthesizing the RNA molecule completely or partially in vitro.
xe2x80x9cNucleotide sequencexe2x80x9d refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide-nucleic acid sequences that may be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or partially synthesized by combining shorter oligonucleotide linkers, or from a series of oligonucleotides, to provide a sequence which is capable of specifically binding to an mRNA molecule and acting as an antisense construct to alter, reduce, or inhibit the transcription of the message into polypeptide, and thus, ultimately affect the concentration, amount, or activity of the final gene product in situ, in vitro, or in vivo.